Back to EveryPatent.com
| United States Patent |
5,210,263
|
|
Kozikowski
, et al.
|
May 11, 1993
|
Inositol phosphate analogs and methods for their use
Abstract
The present invention provides novel inositol phosphate analogs and a
method for their use for detecting the phosphatidylinositol
cycle-dependent calcium of a cell.
| Inventors:
|
Kozikowski; Alan P. (Ponte Verda Beach, FL);
Powis; Garth (Tucson, AZ)
|
| Assignee:
|
University of Pittsburgh (Pittsburgh, PA);
Mayo Foundation For Medical Education and Research (Rochester, MN)
|
| Appl. No.:
|
524267 |
| Filed:
|
May 15, 1990 |
| Current U.S. Class: |
558/161; 558/190; 558/192; 558/194 |
| Intern'l Class: |
C07F 009/117 |
| Field of Search: |
558/161,194,192,190
|
References Cited
Other References
Kozikowski, A. P. et al. J. Chem. Soc. Chem. Commun. 1988, 1301-1303.
Kozikowski, A. P. et al. Tetrahedron Lett. 1989, 30(26), 3365-3368.
|
Primary Examiner: Lee; Mary C.
Assistant Examiner: Ambrose; Michael G.
Attorney, Agent or Firm: Pennie & Edmonds
Claims
What is claimed is:
1. A compound represented by the general formula I:
##STR5##
wherein: R.sup.1 is selected from the group consisting of --OP).sub.3
H.sub.2 and --OPO.sub.3.sup..dbd. ;
each R.sup.2 is independently selected from the group consisting of
C.sub.1-8 linear or branched alkyl, --F, --Cl, --Br. --I, NH.sub.2,
--N.sub.3, --NHOH, --NHNH.sub.2, --CN, --NC, --SH, --SeH, --H, and each
R.sup.2 can be joined to form .dbd.NOH, .dbd.NNH.sub.2,
each R.sup.3 is independently selected from the group consisting of
--OPO.sub.3 H.sub.2, --OPO.sub.3.sup..dbd. and --OH;
with the proviso that at least one of said R.sup.2 is H except when:
(i) each R.sup.2 is joined to form .dbd.NOH, .dbd.NNH.sub.2, .dbd.O or
.dbd.S; and
(ii) R.sup.2 is --F, --Cl, --Br, or --I, then the other R.sup.2 is --F,
--Cl, --Br, --I, or H.
2. The compound of claim 1 wherein R.sup.1 and each R.sup.3 are
--OPO.sub.3.sup.50 .
3. The compound of claim 1 of the myo-inositol stereochemistry.
4. The compound of claim 1 wherein at least one of said R.sup.2 is selected
from the group consisting of --F, --Cl, --Br, and --I.
5. The compound of claim 1 wherein at least one of said R.sup.2 is --F.
Description
TABLE OF CONTENTS
1. Technical Field
2. Background of the Invention
3. Summary of the Invention
4. Detailed Description of the Invention
4.1 The Method of the Invention
5. Example
1. TECHNICAL FIELD
The present invention provides novel inositol phosphate analogs and a
method for their use for detecting the phosphatidylinositol
cycle-dependent calcium of a cell.
2. BACKGROUND OF THE INVENTION
For a cell to survive, it must be able to respond rapidly to changes in its
environment. Furthermore, for cells to reproduce and carry out other
co-operative functions, they must be able to communicate efficiently with
each other. Cells most frequently adapt to their environment and
communicate with one another by means of chemical signals. An important
feature of these signaling mechanisms is that in almost all cases a cell
is able to detect a chemical signal without it being necessary for the
chemical messenger itself to enter the cell. This permits the cell to
maintain tight control of its internal milieu, thereby permitting the cell
to respond to its environment without being destroyed by it.
These sensing functions are carried out by a variety of receptors, which
are dispersed on the outer surface of the cell and function as molecular
antennae. These receptors detect an incoming messenger and activate a
signal pathway that ultimately regulates a cellar process such as
secretion, contraction, metabolism or growth. The major barrier to the
flow of information is the cell's cellular plasma membrane, where
transduction mechanisms translate external signals into internal signals,
which are then carried throughout the interior of the cell by "second
messengers."
In molecular terms, the process depends on a series of proteins within the
cellular plasma membrane, each of which transmits information by inducing
a conformational change--an alteration in shape therefore, in function--in
the protein next in line. At some point the information is assigned to
small molecules or even to ions within the cell's cytoplasm, which serve
as the above-mentioned second messengers, whose diffusion enables a signal
to propagate rapidly throughout the cell.
The number of second messengers appears at present to be surprisingly
small. To put it another way, the internal signal pathways in cells are
remarkably universal, and have been phylogenetically preserved over
millions of years of evolution. Yet the known messengers are capable of
regulating a vast variety of physiological and biochemical processes. The
discovery of the identity of particular second-messenger substances is
proving, therefore, to be of fundamental importance for understanding how
cellular growth and function are regulated.
Several major signal pathways are now known, but two seem to be of primary
importance. One employs cyclic nucleotides as second-messengers. These
cyclic nucleotides activate a number of proteins inside the cell, which
then cause a specific cellular response. The other major pathway employs a
combination of second messengers that includes calcium ions as well as two
substances whose origin is remarkable: inositol 1, 4, 5 trisphosphate
(IP.sub.3) and diacylglycerol (DG). These compounds are released from the
plasma membrane itself, by enzymes that are activated by specific cellular
membrane receptors. However, it should be noted that myo-inositol in its
non-phosphorylated form first enters an organism through the organism's
diet, but can then be recycled as described hereinbelow.
IP.sub.3 is formed by the following scheme. A receptor molecule on the
surface of the cellular plasma membrane transmits information through the
cellular plasma membrane and into the cell by means of a family of G
proteins, which are cellular plasma membrane proteins that cannot be
active unless they bind to guanosine triphosphate (GTP). The G proteins
activate the so-called "amplifier" enzyme phospholipase C, which is on the
inner surface of the cellular plasma membrane. Phospholipase C cleaves the
cellular plasma membrane lipid phosphatidylinositol 4, 5-bisphosphate
(PIP.sub.2) into DG and IP.sub.3. IP.sub.3 is a water-soluble molecule
and, therefore, upon being released from the inner surface of the cellular
plasma membrane, it rapidly diffuses into the cytoplasm. IP.sub.3 then
releases calcium from internal compartments, which store high
concentrations of calcium. The calcium released by IP.sub.3 in turn
activates a large number of intracellular enzymes that orchestrate a
complex set of responses that allow the cell to adapt to the original
signal triggering the receptor that caused the release of IP.sub.3.
Quite fascinatingly, DG and IP.sub.3 are recycled. DG is recycled by a
series of chemical reactions which constitute one component of the lipid
cycle. IP.sub.3 is recycled by a series of reactions known as the
phosphatidylinositol cycle. The two cycles converge at the point when
inositol is chemically linked to DG. The DG-bound inositol is
phosphorylated in a series of steps which ultimately results in the
resynthesis of phosphatidylinositol bisphosphate.
In the first portion of the lipid cycle, DG is converted to phosphatidic
acid, which in turn is converted to cytidine diphosphate diglyceride
(CDP-DG), while in the first portion of the phosphatidylinositol cycle,
IP.sub.3 is dephosphorylated to ultimately form myoinositol. It is
believed that such dephosphorylation occurs stepwise; IP.sub.3 is
converted to an inositol bearing only two phosphate groups (IP.sub.2),
followed by the loss of an additional phosphate, resulting in IP.sub.1,
which is then dephosphorylated to myo-inositol. Also, it has been shown
that IP.sub.3 can also undergo an additional phosphorylation, thereby
being converted to inositol 1, 3, 4, 5 tetrakisphosphate (IP.sub.4). This
molecule is subsequently metabolized by successive removal of phosphate
groups, as described above. It is believed that phosphatase enzymes
catalyses each step of this process.
The lipid cycle and phosphatidylinositol cycle merge by the myo-inositol
reacting with the CDP-DG to form phosphatidylinositol (PI) PI is
phosphorylated to ultimately form PIP.sub.2. It is believed that such
phosphorylation occurs stepwise; PI is converted to phosphatidyl
myo-inositol 4-phosphate (PIP), which is converted to PIP.sub.2 ; the
final step of both cycles. It is believed that a kinase enzyme catalyses
each step of this process.
For an excellent review of IP.sub.3, its role as a second messenger and the
phosphatidylinositol cycle see Berridge, M., et al. Inositol Triphosphate,
a Novel Second Messenger in Cellular Signal Transduction, Nature, 312,
315-321 (1984) and Berridge, M. The Molecular Basis of Communication
Within the Cell, Scientific American, 142-152 (October 1985), and James W.
Putney, Jr. (Ed.), Phosphoinositide and Receptor Mechanisms, Alan R. Liss,
Inc., New York, N.Y. 1986.
3. SUMMARY OF THE INVENTION
The present invention relates to a method for detecting the
phosphatidylinositol cycle-dependent calcium of a cell comprising:
a) contacting said cell with a compound represented by general formula I
thereby permitting said compound to enter said cell:
##STR1##
wherein:
R.sup.1 is selected from the group consisting of --OPO.sub.3 H.sub.2 and
--OPO.sub.3.sup.50 ;
each R.sup.2 is independently selected from the group consisting of
C.sub.1-8 linear or branched alkyl, --F --Cl, --Br, --I, --NH.sub.2,
--N.sub.3, --NHOH, --NHNH.sub.2, --CN, --NC, --SH, --SeH, --H, and each
R.sup.2 can be joined to form .dbd.NOH, .dbd.NNH.sub.2, .dbd.O or .dbd.S;
and
each R.sup.3 is independently selected from the group consisting
--OPO.sub.3 H.sub.2, --OPO.sub.3.sup.50 and --OH;
with the proviso that at least one of said R.sup.2 is H except when:
(i) each R.sup.2 is joined to form .dbd.NOH, .dbd.NNH.sub.2, .dbd.O or
.dbd.S; and
(ii) R.sup.2 is --F, --Cl, --Br, or --I, then the other R.sup.2 is --F,
--Cl, --Br, --I, or H; and
b) detecting the calcium of said cell.
The present invention also provides the compounds represented by general
formula I.
DETAILED DESCRIPTION OF THE INVENTION
4 1 The Method of the Invention
The present invention relates to a method for detecting the
phosphatidylinositol cycle-dependent calcium of a cell comprising:
a) contacting said cell with a compound represented by general formula I
thereby permitting said compound to enter said cell:
##STR2##
wherein:
R.sup.1 is selected from the group consisting of --OPO.sub.3 H.sub.2 and
--OPO.sub.3.sup..dbd. ;
each R.sup.2 is independently selected from the group consisting of
C.sub.1-8 linear or branched alkyl, --F, --Cl, --Br, --I, --NH.sub.2,
--N.sub.3, --NHOH, --NHNH.sub.2, --CN, --NC, --SH --SeH, --H, and each
R.sup.2 can be joined to form .dbd.NOH, .dbd.NNH.sub.2, .dbd.O or .dbd.S;
and
each R.sup.3 is independently selected from the group consisting of
--OPO.sub.3 H.sub.2, --OPO.sub.3.sup..dbd. and --OH; with the proviso that
at least one of said R.sup.2 is H except when:
(i) each R.sup.2 is joined to form .dbd.NOH, .dbd.NNH2, .dbd.0 or .dbd.S;
and
(ii) R.sup.2 is --F, --Cl, --Br, or --I, then the
other R.sup.2 is --F, --Cl, --Br, --I, or H; and
b) detecting the calcium of said cell.
It should be noted that general formula I, a Haworth projection, depicts
the absolute stereochemistry.
The present invention also provides the compounds represented by general
formula I. In a preferred embodiment, R.sup.1 and each R.sup.3 are
--OPO.sub.3.sup..dbd.. It is also preferred that general formula I have
the myoinositol stereochemistry, i.e. the R.sup.2 that is depicted in the
Haworth projection below the plane of the cyclohexane ring be H.
Without being bound by theory, it is believed that the compounds of general
formula I will mimic the action of IP.sub.3. Thus, the compounds of
general formula I will increase free intracellular calcium levels. This is
because IP.sub.3 mobilizes intracellular calcium and, some theorize, also
permits extracellular calcium to enter the cell. However, unlike IP.sub.3,
the compounds of general formula I will not be converted to IP.sub.4,
thereby avoiding complications due to action of IP.sub.4 on free
intracellular calcium levels. Accordingly, the compounds of general
formula I can be utilized to study IP.sub.3 associated intracellular free
calcium levels. Thus, one can detect cellular phosphatidylinositol
cycle-dependent calcium changes. This is useful, for example, because one
can detect the change in phosphatidylinositol cycle-dependent calcium
caused by pharmacological agents that are designed to work through the
phosphatidylinositol cycle.
The method of the invention is carried out by contacting the cell to be
analyzed with a compound represented by general formula I thereby
permitting the compound to enter the cell. Any type of cell can be
utilized in the method. The choice of cell depends only on the cell system
that one wants to study.
Prior to use, the compound represented by general formula I should be
dissolved in an aqueous medium. The compound represented by general
formula I is then contacted with the cell. It is essential that the
contacting step results in the compound represented general formula I
entering the cell. This contacting step can be carried out by any
technique, for example, selective permeabilization, microinjection or
caging. These three standard techniques are described in Gill, D. L. and
Cheuh, S. H., An intracellular (ATP+Mg.sup.2+) dependent calcium pump
within NlE-115 neuronal cell line, J. Biol. Chem. 260:9289-9297 (1985);
Oakes, S. G., Iaizzo, P. A., Richelson, E. and Powis, G.,
Histamine-induced intracellular free Ca.sup.2+, inositol phosphates and
electrical changes in murine NlE-115 neuroblastoma cells, Pharmacol. Exp.
Ther. 247:114-121 (1988); and Walker, J. W., Somlyo, A. V., Goldman, Y.
E., Somlyo, A. P. and Trentham, D. R., (1987) Kinetics of smooth and
skelatal muscle activation by laser pulse photolysis of caged
inositol-1,4,5-trisphosphate, Nature 327:249-252 (1987 ).
After the contacting step the calcium in the cell can be detected. Such
detection can be carried out by any technique, for example, by utilizing
.sup.45 Ca.sup.+2, calcium sensitive microelectrodes, Ca.sup.+2 sensitive
fluroescent indicators, or Ca.sup.+2 sensitive bioluminescent indicators.
See Gill, D. L. and Cheun, S. H., An intracellular (ATP+Mg.sup.+2 )
dependent calcium pump within N1E-115 neuronal cell line, J. Biol. Chem.,
260:9289-9297 1985; Ammann, D., Meier, P. C. and Simon, W., Design and use
of calcium sensitive microelectrodes, Detection and Measurement of Free
Ca.sup.2+ in Cells, (Eds. C. C. Ashley and A. K. Campbell), pp 117-129,
Elsevier/North Holland, Amsterdam (1979); Grynkiewicz, G. Poenie, M. and
Tsien, R. Y., A new generation of Ca.sup.2 + indicators with greatly
improved flourescence properties, J. Biol. Chem. 260:3340-3450; and
Blinks, J. R., Methods for monitoring Ca.sup.2+ concentrations with
photoproteins in living cardiac cells., Methods for Studying heart
Membranes, Volume II (Ed. N. S. Dhalla), pp. 237-264, CRC Press, Boca
Raton (1984). These techniques also permit one to not only detect the
calcium but also to measure the level of calcium.
In a preferred embodiment of the invention, one can measure the level of
calcium in the cell prior to the contacting step. Thus, this permits one
to detect and measure the change in the phosphatidlylinositol
cycle-dependent calcium. Of course, this measurement of the level of
calcium can be carried out by, for example, the techniques described
hereinabove.
5. SYNTHESIS OF COMPOUNDS OF GENERAL FORMULA I
Methods of Synthesis of D-3-Deoxy-3-Fluoro-myo-Inositol 1,4,5-Trisphosphate
A detailed description of the method of synthesis of the title compound
follows. Formulae for all the structures of the compounds named in the
detailed description can be found in Scheme 1.
##STR3##
Preparation of 1,2:5,6-di-O-cyclohexylidene-3-deoxy-3-fluoro-myo-inositol
(1) and 1,2:4,5-di-O-cyclohexylidene-3-deoxy-3-fluoro-myo-inositol (2)
To a stirred mixture of 3-deoxy-3-fluoro-myo-inositol (1,82 g, 10.0 mmol)
and camphorsulfonic acid (50 mg) in DMF at 55.degree. C. under argon was
addes neat 2-methoxypropene (3.83 mL, 40 mmol). The mixture was stirred
for 4 h at 80.degree. C. and cooled. Triethylamine (4 mL) was added and
the solvent was distilled off in vacuo. The light brown residue was
chromatographed over silica gel using 40-50% ethyl acetate in hexane to
furnish a 2.3:1 ratio of the diacetonides 1 and 2; yield =2.1 g, 82.6%.
1. [.varies.].sub.D.sup.23 -52.degree. (c=5 mg/mL, CHCl.sub.3): mp
154.degree.-156.degree. C.; IR (Nujol) 3520, 2990, 1373, 1240, 1057, 868
cm.sup.-1 ; .sup.1 H NMR .delta. 4.64 (ddd, J=46.4, 3.3, 3.3 Hz, H-3),
4.95 (ddd, J=22.6, 6.6, 3.3 Hz, H-2), 4.41 (ddd, J=6.6, 6.6 Hz, H-1), 4.27
(ddd, J=17.3, 8.5, 3.4, 3.3 Hz, H-4), 3.98 (ddd, J=10.7, 6.6, 3.3 Hz,
H-6), 3.47 (dd, J=10.7, 8.5 Hz, H-5) 2.44 (d, J=3.4 Hz, OH), 1.53 (s, 3
H), 1.45 (s, 6H, 1.39 (s, 3 H); .sup.13 C NMR (75 MHz, CDCl.sub.3)
.delta.112.62 (s), 110.31 (s), 88.25 (d,J=189 Hz), 81.41 (s), 76.81,
75.81, 75.19 (d, J=15 Hz), 74.72 (d, J =18.7 Hz), 73.64 (s), 27.48 (s),
26.30 (s), 26.29 (s), 25.37 (s); mass spectrum, (EI) m/z 247 (M.sup.+
-CH.sub.3), 207, 187, 101, 59; HRMS calcd for M.sup.+ -CH.sub.3 247.0982,
found 247.0982.
2. [.varies.].sub.D.sup.23 +7.2.degree. (c=5 mg/mL, CHCl.sub.3); mp
206.degree.-207.degree. C.; IR (KBr disk) 3550, 2980, 1773, 1238, 1050,
867 ch.sup.-1 ; .sup.1 H NMR (300 MHz, CDCl.sub.3) .delta. 4.86 (ddd,
J=48.8, 10.1, 4.6 Hz, H-3), 4.78 (ddd, J=4.6, 2.3, 2.3 Hz, H-2), 4.14 (dd,
J=10.1, 10.1 Hz, H-4), 4.08 (dd, J=6.5, 2.3 Hz, H-1), 3.94 (ddd, J=10.6,
6.5, 3.0 Hz, H-6), 3.33 (ddd, J=10.6, 10.1 1.5 Hz, H-5), 2.63 (d, J=3.0
Hz, OH), 1.57 (s, 3H), 1.49 (s, 3 H), 1.47 (s, 3 H), 1.40 (s, 3H); .sup.13
C NMR (75 MHz, CDCl.sub.3) .delta. 112.04 (s), 110.79 (s), 93.64 (d, J=187
Hz), 77.78 (s), 76.89 (s), 75.97 (s), 73.91 (d, J=18 Hz), 71.0 (d, J=2.5
Hz), 26.45 (s, 2 C),, 26.15 (s), 24.79 (s); mass spectrum, (EI) m/z 262
(M.sup.+) , 247, 129, 101, 59; HRMS calcd for M.sup.+ -15 247.0982, found
247.0982.
Preparation of
6-O-benyl-1,2:4,5-di-O-cyclohexylidene-3-deoxy-3-fluoro-myo-inositol (3)
To a stirred suspension of sodium hydride (0.5 g of 50% oil dispersion) in
20 mL of dry THF under argon, was added a solution of the diacetonide 2
(1.7 g, 6.5 mmol) in THF via cannula. After stirring for 15 min, a
solution of benzyl bromide (1.24 mL, l0.41 mmol) in THF was added via
cannula. The mixture was stirred overnight and quenched by adding water
and ether. The aqueous phase was extracted with ether three times. The
ether extracts were combined and washed with water and saturated sodium
chloride and dried (MgSO.sub.4). After concentration in vacuo, a glassy
solid was obtained which was chromatographed on silica gel (20% ethyl
acetate in hexane) to yield 2.19 g (96%) of the diacetonide 3:
[.varies.].sub.D.sup.23 -79.7.degree. (c=7.9 mg/mL, CHCl.sub.3); mp
135.degree.-138.degree. C.; IR (Nujol) 2990, 2950, 1575, 1375, 1220, 1080
cm.sup.-1 ; .sup.1 H MHz, CDCl.sub.3) .delta.7.40-7.25 (m, 5 H), 4.81
(ddd, J=49, 10.2, 4.5 Hz, H-3), 4.55 (ddd, J=4.6, 2.4, 2.4 Hz, H-2), 4.20
(dd, J=5.8, 5.8 Hz, H-1), 4.09 (dt, J=19.4, 9.7, 9.7, H-4), 3.70 (dd,
J=10.8, 6.5, H-5), 3.40 (ddd, J=10.8, 10.8, 1.5 Hz, H-6), 1.49 (s, 3 H),
1.46 (s, 3 H), 1.39 (s, 3 H), 1.37 (s, 3 H); .sup.13 C NMR (75 Hz,
CDCl.sub.3) .delta.137.5 (s), 127.73 (s, 2 C), 127.44 (s, 2 C), 127.08
(s), 112.39 (s), 110.09 (s), 88.22 (d, J=188 Hz), 80.81 (s), 79.20 (s),
77.25 (s), 75.27 (d, J=15 Hz), 74.62 (d, J=18 Hz), 71.50 (s), 27.20 (s),
26.35 (s), 26.34 (s), 25.39 (s); mass spectrum, (EI) m/z 352 (M+), 337
(M+-CH3), 294, 246, 203, 145, 91, 59; HRMS calcd for C.sub.19 H.sub.25
FO.sub.5 352.1696, found 352.1686.
Preparation of
6-O-benzyl-1,2-O-cyclohexylidene-3-deoxy-3-flouro-myo-inositol (4)
A solution of the dicetonide 3 (2.0 g, 5. mmol) and four drops of acetyl
chloride in 100 mL of methanol was stirred at room temperature for 4 h or
until the TLC showed completion of reaction. One mL of triethylamine was
added and the volatiles were evaporated under reduced pressure and the
pale yellow residue was chromatographed (silica gel) with 80% ethyl
acetate in hexane to furnish 1.53 g (86%) of the monoacetonide 4 as a
solid.
[.varies.].sub.D.sup.23 -30.4.degree. (c=4.5 mg/mL, MeOH); mp
141.degree.-144.degree. C.; IR (Nu1ol) 3566, 3250, 1366, 1105, 870
cm.sup.-l ; .sup.1 H NMR (300 MHz, CDCl.sub.3) .delta.6 4.93 (d, J=11.5
Hz, --CH.sub.2 --), 4.66 (d, J=1.5 Hz, --CH.sub.2 --), 4.60 (ddd, J=47,
8.6, 4.7 Hz, H-3), 4.52 (m, H-2), 4.24 (dd, J=6.6, 5.8, H-1), 4.08 (m,
H-4), 3.59 (dd, J=9.5 6.6, H-6), 3.41 (ddd, J=9.5, 9.5, 2.4 Hz, H-5), 2.79
(m, 2 OH), 1.52 (s, 3 H), 1.40 Is, 3 H); .sup.13 C NMR (75 MHz,
CDCl.sub.3) .delta. 137.75 (s), 128.50 (s), 128.08 (s), 127.99 (s), 10.8
(s), 90.3 (d, J =184 HZ), 81.33 (s), 79.97 (s), 74.10 (d, J=15.7 Hz),
73.25, 72.50 (d, J=9.8 Hz), 70.82 (d, J=21 Hz), 27.67 (s), 25.84 (s); mass
spectrum, (EI) m/z 312 (M+), 297, 254, 173, 107, 91; HRMS calcd for
C.sub.16 H.sub.21 FO.sub.5 312.1373, found 312.1373.
Preparation of
1,2-O-oyclohexylidene-4,5-di-0-benzoyl-6-O-benzyl-3-deoxy-3-fluoro-myo-ino
sitol (5)
Benzoyl chloride (1.9 mL, 16.0 mmol) was added via syringe under argon, to
a stirred solution of the 5 diol 4 (2.0 g, 6.4 mmol) in 60 mL of pyridine
at room temperature. After stirring for 12 h pryidine was distilled off in
vacuo and the light yellow residue was directly chromatographed on silica
gel using 20% ethyl acetate in hexane to afford 3.06 g (92%) of the
dibenzoate 5 as a foam.
[.varies.].sub.D.sup.23 -52.24.degree. (c=12.25 mg/mL, CHCl.sub.3); mp
below 70.degree. C.; IR (Nujol) 1728, 1425, 1275, 1100, 1070 cm.sup.-1 ;
.sup.1 H NMR (CDCl.sub.3, 300 MHz) .delta. 8.00 (d, J=7.7 Hz, 4 H), 7.51
(m, 2 H), 7.40 (m, 4 H), 7.25 (m, 5 H), 5.98 (dd, J=16.8, 9.3, H-4), 5.49
(dd, J=6.8, 6.8 Hz, H-2), 5.06 (ddd, J=47, 9.3, 3.7 Hz, H-3), 4.85 (bs,
--CH.sub.2 --), 4.73 (m, H-2), 4.5 (dd, J=6.6 Hz, H-6), 3.98 (dd, J=6.8, 6
Hz, H-1), 1.63 (s, 3 H), 1.41 (s, 3 H); .sup.13 C NMR (CDCl.sub.3, 75 MHz)
.delta. 165.77 (s), 165.42 (s), 137.34 (s), 133.26 (s), 129.84 (s),
129.40 (s), 129.27 (s), 128.35 (s), 127.96 (s), 127.86 (s), 110.98 (s),
87.60 (d, J=187 Hz), 77.51 (d, J=14.2 Hz), 76.69 (d, J=31.2 Hz), 73.44 (m,
2 C), 72.76 (s), 70.71 (d, J=21.8 Hz), 26.79 (s), 24.89 (s); mass
spectrum, (El) m/z 505 (M+-15), 415, 309, 292, 277, 105, 91; HRMS calcd
for C.sub.29 H.sub.26 FO.sub.7 505.1663, found 505.1663.
Preparation of 4,5-di-O-benzoyl-6)-benzyl-3deoxy-3-fluore-myo-inositol (6)
The dibenzoate 5 (3.2 g, 6.1 mmol) was dissolved in 100 mL of MeOH
containing 20 drops of conc. HCI. The mixture was let stand at room
temperature for 6 h. Triethylamine (4 mL) was added and the volatiles were
stripped off under vacuum. The resulting residue was directly
chromatrographed on silica gel using 40% ethyl acetate in hexane to yield
2.7 g (92%) of the diol 6 as a white solid.
[.varies.].sub.D.sup.23 -65.8. (c=4.6 mg/mL, CHCl.sub.3); mp
64.degree.-66.degree. C.; IR (thin film) 3500, 1728, 1718, 1450, 1277,
1105, cm.sup.-1 ; .sup.1 H NMR (CDCl.sub.3, 300 MHz) .delta.7.90 (m, 4 H),
7.49 (m, 2 H), 7.38 (m, 4H), 7.19 (m, 5 H), 6.06 (dd, J=21, 10 Hz, H-4),
5.55 (dd, J=10, 10 Hz, H-5), 4.72 (ddd, J=47.7, 9.7, 2.8 Hz, H-3), 4.69
(AB q, J=11.2 Hz, --CH.sub.2 --) 4.48 (m, H-2), 4.13 (dd, J=9.5, 9.5 Hz,
H-6), 3.82 (m, H-1), 2.77 (bs, OH), 2.59 (d, J=5 Hz, OH); .sup.13 C NMR
(CDCl.sub.3, 75 MHz) .delta.165.71 (s), 137.51 (s), 133.25 (s), 133.20
(s), 129.75 (s), 129.12 (s), 128.39 (s), 128.30 (s), 128.05 (s), 127.92
(s), 89 9 (d, J=187 Hz), 79.10 (s), 75.31 (s), 71.95 (d, J=12.4 Hz), 70.85
(s), 70.60 (d, J=10.1 Hz), 70.15 (d, J=17.3 Hz); mass spectrum, (EI) mz/
480, 375, 269, 252, 105, 91, 77; HRMS calcd for C.sub.27 H.sub.25 FO.sub.7
480.15843, found 480.15843.
Preparation of 1,4,5-tri-O-benzoyl-6-O-benzyl-3-deoxy-3-fluoro-myo-inositol
(7)
A mixture of the diol 6 (0.63 g, 1.32 mmol), 169 mL of benzoyl chloride,
and a few crystals of DMAP in 20 mL of dry pyridine under argon was
stirred for 8 h at 0.degree. C. Pyridine was distilled off under vaccum,
and the yellow residue was directly purified by silica gel chromatography
using a gradient elution (40%-60% ethyl acetate in hexane) to give 0.692 g
(89.3%) of the tribenzoate 7 as a white solid.
[.varies.].sub.D.sup.23 -52.6.degree. (c=3.65 mg/mL, CHCl.sub.3); mp
180.degree.-183.degree. C.; IR(Nujol) 3500, 1728, 1452, 1315, 1271, 1107,
1028 cm.sup.-1 ; .sup.1 H NMR (CDCl.sub.3, 300 MHz) .delta.8.09 (d, J=7.3
Hz, 2 H), 7.93 (t, J=8.7 Hz, 4 H), 7.61 (t,J=7.5 Hz), 7.48 (m, 4 H), 7.36
(t, J=7.7 Hz, 4 H), 7.01 (m, 5 H), 6.13 (dd, J=21, 10 Hz, H-4), 5.66 (dd,
J=9.8, 9.8 Hz, H-5), 5.29 (bd-, J=9.8 Hz, H-1), 4.83 (ddd, J=48, 9.5, 2.5
Hz, H-3), 4.72 (m, H-2), 4.65 (AB q, J=11.2 Hz, --CH.sub.2 --), 4.53 (dd,
J=9.8, 9.8 Hz, H-6), 2.77 (--OH); .sup.13 C NMR (CDCl.sub.3, 75 MHz)
.delta.165.61(s), 133.51(s), 165.42 (s), 133.18(s), 129.81 (s), 129.74
(s), 129.28 (s), 129.15 (s), 129.08 (s), 128.54 (s), 128.31 (s), 128.11
(s), 127.89 (s), 127.63 (s), 89.74 (d, J=185 Hz), 75.35 (s), 72.62 (d,
J=11.2 Hz), 71.72 (d, J=12.8 Hz), 70.50 (d, J=20 Hz), 68.7 (d, J=19.5 Hz);
mass spectrum, (EI) m/z 584 (M+), 566, 479, 176, 106, 91; HRMS calcd for
C.sub.34 H.sub.29 FO.sub.8 584.1675, found 584.1675.
Preparation of
1,4,5-tri-O-benzoyl-6-O-benzyl-3-deoxy-3-fluoro-myo-inositol, 2-ethoxyethy
l ether (8)
A homogenous solution of the tribenzoate 7 (0.692 g, 1.18 mmol), ethyl
vinyl ether (140 .mu.L, 1.56 mmol) and a catalytic amount of pyridium
p-toluenesulfonate was stirred at room temperature in CH.sub.2 Cl.sub.2
for 12 h. Solvent was removed under reduced pressure and the resulting
residue was chromatographed on silica gel to afford 0.67 g (86.2%) of the
protected tribenzoate 8 as a foam.
[.varies.].sub.D.sup.23 -45.degree. (c=1.8 mg/mL, CHCl.sub.3); mp
65.degree.-68.degree. C.; IR (thin film) 1728, 1452, 1315, 1269, 1176,
1095, 960 cm.sup.-1 ; .sup.1 H NMR (CDCl.sub.3, 300 MHz) .delta.8.05 (m, 2
H), 7.61 (m, 1 H), 7.50 (m, 4 H), 7.39 (m, 4 H), 7.10 (m, 5 H), 6.2-6.0
(m, 1 H), 5.63 (t, J=9.8 Hz, 1 H), 5.22 (m, 1 H), 4,90-4.40 (m, 5 H), 3.91
(m, 5 H), 3.65-3.51 M, 1.5 H), 3.32 (m, 1 H), 1.47 (d, J=5.2 Hz, 1.5 H),
I.32 (d, J=5 Hz, 1.5 H), 1.19 (t, J=7 Hz, 1.5 H), 0.80 (t, J=7 Hz, 1.5 H);
mass spectrum, (EI) m/z 656 (M ), 585, 408,372,208,190,91; HRMS calcd for
C 656.7113, found 656.7113.
Preparation of 6-O-benzyl-3-deoxy-3-fluoro-myo-inositol, 2-ethoxyethyl
ether (9)
Anhydrous potassium carbonate (2.0 g. 14.4 mmol) was added to a stirred
solution of the tribenzoate 8 (0.67 g, 1.02 mmol) in 50 mL of anhydrous
methanol. The suspension was stirred overnight at room temperature and
concentrated at reduced pressure. Water and NaCl were added and the
aqueous phase was extracted with ethyl acetate(3.times.). After drying
(MgSO.sub.4), and concentration, the residue was purified by silica gel
chromatography (70% ethyl acetate in hexane) to leave 0.29 g (835) of the
triol as a wax.
[.varies.].sub.D.sup.23 -11.16.degree. (c=28 mg/mL, CHCl.sub.3); IR (thin
film) 3475, 1475, 1375, 1105, 1050 cm.sup.-1 ; .sup.1 H NMR (CDCL.sub.3,
300 MHZ) 6 740-7.24 (m, 5 H), 5.06 (d, J=11.5 Hz, 0.5 H), 4.86 (m, 0.5 H),
4.81 (AB q, J=11.2 Hz, I H), 4.69 (d, J=11.2 Hz, 0.5 H), 4.62 (m, 0.5 H),
4.40 (m, 0.5 H), 4.21 (m, 1 H), 4.18-3.95 (m, 1.5 H), 3.82 (m, 1 H),
3.62-4.47 (m, 3 H), 3.41 (m, 0.5 H), 3.31 (m, 0.5 H), 2.8 (bs, 3 OH), 1.38
(d, J=5 Hz, 1.5 H), 1.32 (d, J=5 Hz, 1.5 H), 1.22 (t, J=7 Hz, 1.5 H),
1.21 (t, J=7 Hz, 1.5 H); mass spectrum (EI) m/z 344 (M ), 326, 308, 244,
189, 91; HRMS calcd for C.sub.17 H.sub.25 FO.sub.6 344.2941, found
344.2941.
Preparation of 6-O-benzyl-3-deoxy-3-fluoro-myo-inositol
1,4,5-trisphosphate, hexabenzyl ester (11)
Sodium hydride (0.179 g, 3.7 mmol, 50% suspension in mineral oil) was added
under argon to a stirred solution of the triol (o.117 g, 0.34 mmol) and
tetrabenzyl pyrophosphate (1.10 g, 2.04 mmol) in anhydrous DMF at
0.degree. C. After stirring the mixture between 0.degree.-5.degree. C. for
9 h, the DMF was distilled off at 0.5 mm Hg pressure using a cold
(25.degree. C.) water bath. Methylene chloride (50 mL) was added, and the
heterogeneous suspension was stirred for 15 min and filtered through
Celite. The fitrate was concentrated in vacuo. The white residue was
directly chromatographed on silica gel (60% ethyl acetate in hexane) to
yield 0.372 g (95%) of 10 as a colorless oil. (FAB) 1125 (M.sup.+ 1),
1079, 1053, 963, 873, 419, 391, 181, 149, 129.
A few crystals of p-toluenesulfonic acid were added to a stirred solution
of 10 (0.36 g, 0.32 mmol) in 10 mL of absolute methanol for 3h. Methanol
was stripped off under rotary evaporation, and the resulting residue was
rapidly filtered through silica gel with 80% ethyl acetate in hexane to
afford 0.28 g (78.2%) of the 11 as a wax.
[.varies.].sub.D.sup.23 -6.8.degree. (c =30 mg/mL, CHCl.sub.3); IR (thin
film) 3300, 1496, 1456, 1381, 1271, 1155, 1016 cm.sup.-1 ; .sup.1 H NMR
(CDCl.sub.3, 300 MHz) .delta. 7.40-6.90 (m, 35 H), 5.15-4.68 (m, 14 H),
4.63 (dd, J=11.7 Hz, 9 Hz, 1 H), 4.50-4.37 (m, 2.5 H, includes a part of
the signal for C-3 proton), 4.21 (dd, J=9.5, 2.3 Hz, 0.5 H, a part of
C-3), 4.15 (bt, J=8.4 Hz, 1 H), 3.9 (t, J=9.4 Hz, 1 H), 2,99 (bs, OH);
.sup.31 P NMR (CDCl.sub.3, 201 MHz, 85% phosphoric acid standard,
H-decoupled) .delta.-4.34, -4.79, -5.00 ppm; .sup.19 F NMR (470 MHz,
CDCl.sub.3, CFCl.sub.3 as standard, .sup. 1 H-coupled) .phi.204.57 (dt,
J=47, 9.9 Hz); (FAB) 1053 (M.sup.+ +1), 963, 873, 783, 181, 136.
Preparation of 3-deoxy-3-fluoro-myo-inositol-1,4,5trisphosphate, hexasodium
salt (12)
A 25 mL round-bottomed flask was charged with 11 (0.28 g, 0.266 mmol) and
15 mL of absolute ethanol. Platinum oxide (20 mg) was added, and the
contents were purged with hydrogen and stirred in the same atmosphere
(using a balloon) at room temperature overnight. Following filtration over
Celite and concentration in vacuo, the oily residue was dissolved in 2 mL
of distilled water and treated with 6 equiv. of 1N NaOH and concentrated
to a small (1 mL) volume Methanol (5 mL) was added, and the white
precipitate formed was filtered and washed several times with methanol and
dried to afford 0.12 g (79%) of 12 as a white powder.
[.varies.].sub.D.sup.23 -8.5.degree. (c=3.75 mg/mL, H.sub.2 O); .sup.1 H
NMR (D.sub.2 O, 500 MHz, pH=9, D.sub.2 O peak set at 4.78 ppm) .delta.4.56
(m, 1.5 H, includes a part of the signal for C-3 proton), 4.50-4.36 (m,
1.5 H, includes a part of the signal for C-3 proton), 3.920-3.78 (m, 3 H);
.sup.31 P NMR (D.sub.2 O, pH=9, 201 MHz, H-coupled) .delta.4.63 (bs), 3.38
(bs), 319(s); .sup.19 F NMR (D.sub.2 O, pH=9, 470 MHz, H-coupled)
.PHI.198.02 (bd, J.about.44 Hz).
By employing one of the other halogen (Br, Cl or I) substituted inositols
in place of D-3-deoxy-3-fluoro-myo-inositol in the above reaction
sequence, any of the other 3-halogen substituted inositol
1,4,5-trisphosphates can be prepared. To prepare the 1,4bisphosphate,
1,5-bisphosphate or 1-phosphate analogues, protection of either the
4-hydroxyl group or the 5-hydroxyl group, or both hydroxyl groups by
benzylation is required. After phosphorylation of any remaining free
hydroxyl groups as in the conversion of 9 to 10 in the above scheme, all
benzyl groups are removed by hydrogenolysis and any remaining protecting
groups cleaved under standard conditions to provide the desired
monophosphate or bisphosphate analogues.
To prepare the inositol 1,4,5-trisphosphate analogues bearing an amino or
azido group at the D-3 position, the following sequence of synthetic
operations can be employed (Scheme 2). In view of the extensive
experimental description provided for the synthesis of
D-3-deoxy-3-fluoro-myo-inositol 1,4,5-trisphosphate only the chemical
reaction sequence is drawn out for the amine and azide bearing inositol
phosphates. The methods-used involve standard synthetic operations. By
starting from other substituted inositols (e.g., --SH, CN,--N=C, etc.) and
following similar reactions schemes, any of the other inositol phosphates
of the present invention can be prepared readily.
##STR4##
EXAMPLE
To investigate the effect of 3-F IP.sub.3 on Ca.sup.2+ release, Swiss 3T3
cells were permeabilized with medium containing 0.005% saponin as
described in Seewald et al., Cancer Communications, 1,151 (1989). After
washing, the cells were incubated in medium containing 1 mM ATP, 3%
polyethlene glycol, 50 uM .sup.45 Ca.sup.2+ (160 uCi/mmol) and EGTA to
buffer the free Ca.sup.2+ to a concentration of 10.sup.-7 M. The cells
were collected on glass microfiber filters and washed with buffer
containing 1 mM LaCl.sub.3 prior to liquid scintillation counting.
Preliminary studies showed that .sup.45 Ca.sup.2+ uptake by Swiss 3T3
cells reached a plateau by 6 min. IP.sub.3 (Molecular Probes, Irvine, CA)
or 3-F IP.sub.3 was added at 6.25 min, and the .sup.45 Ca.sup.2+
remaining in the cells was measured at 7 min. All determinations were
conducted in quintuplet..sup.45 Ca.sup. 2+ release is expressed as the
percent release measured at 7 min compared to the 6 min value for each
pair of determinations and corrected for .sup.45 ca.sup.2+ release in the
absence of added agent.
As can be seen from the dose response curve presented in Table I, 3-F
IP.sub.3 acts as a full agonist in releasing .sup.45 Ca.sup.2+ from the
3T3 cells. The unnatural fluorinated IP.sub.3 analogue is equipotent to
natural IP.sub.3. Dextran sulfate, a potent blocker of the release of
Ca.sup.2+ IP.sub.3 also blocked the release of Ca.sup.2+ induced by 3-F
IP.sub.3 (data not shown). From the studies it is apparent that
interaction of IP.sub.3 with its receptor on the edoplasmic reticulum does
not require the 3-hydroxyl group either for recognition or for functional
activity.
The findings obtained from this study are significant, for they reveal an
important new tool for the study of PI-based cell signalling: 3-F IP.sub.3
exhibits the same agonist effects as IP.sub.3 on Ca.sup.2+ release, but
its role is not further complicated by a possible simulatenous action of
3kinase(s) to produce IP.sub.4. This compound is thus to be recommended in
place of IP.sub.3 in studying intracellular Ca.sup.2+ release.
TABLE I
______________________________________
Release of .sup.45 Ca.sup.2+ from non-mitochondrial stores of
saponin-permeabilized Swiss 3T3 cells by 3-F IP.sub.3 and
IP.sub.3.
Concentration (.mu.M)
0 0.5 1.0 3.0 10.0
______________________________________
.sup.45 Ca.sup.2+ release %
IP.sub.3
0 16.1 .-+. 5.2
23.4 .-+. 4.4
31.3 .-+. 4.6
38.2 .-+. 3.7
3-FIP.sub.3
0 12.8 .-+. 5.9
20.1 .-+. 4.3
29.4 .-+. 5.9
39.2 .-+. 2.1
______________________________________
Note: All determinations were conducted in quintuplet
Values are mean .-+. S.D. and are the release at 7 min. expressed as a
percent of the .sup.45 Ca.sup.2+ in the cell at 6 min. (equilibrium
value) corrected for .sup.45 Ca.sup.2+ release in the abscence of added
agents. The agents were added at the concentrations shown at 6.25 min.
Top